Bottom Line:
We show that both the primary transcripts for miR-17 approximately 92 and miR-106b approximately 25 and the pivotal miRNAs that are derived from miR-17 approximately 92 display increased abundance in Toxoplasma-infected primary human cells; a Toxoplasma-dependent up-regulation of the miR-17 approximately 92 promoter is at least partly responsible for this increase.Altered levels of miR-17 approximately 92 and miR-106b approximately 25 are known to play crucial roles in mammalian cell regulation and have been implicated in numerous hyperproliferative diseases although the mechanisms driving their altered expression are unknown.Hence, in addition to the implications of these findings on the host-pathogen interaction, Toxoplasma may represent a powerful probe for understanding the normal mechanisms that regulate the levels of key host miRNAs.

Affiliation: Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT

Background: The apicomplexan parasite Toxoplasma gondii can infect and replicate in virtually any nucleated cell in many species of warm-blooded animals; thus, it has evolved the ability to exploit well-conserved biological processes common to its diverse hosts. Here we have investigated whether Toxoplasma modulates the levels of host microRNAs (miRNAs) during infection.

Methodology/principal findings: Using microarray profiling and a combination of conventional molecular approaches we report that Toxoplasma specifically modulates the expression of important host microRNAs during infection. We show that both the primary transcripts for miR-17 approximately 92 and miR-106b approximately 25 and the pivotal miRNAs that are derived from miR-17 approximately 92 display increased abundance in Toxoplasma-infected primary human cells; a Toxoplasma-dependent up-regulation of the miR-17 approximately 92 promoter is at least partly responsible for this increase. The abundance of mature miR-17 family members, which are derived from these two miRNA clusters, remains unchanged in host cells infected with the closely related apicomplexan Neospora caninum; thus, the Toxoplasma-induced increase in their abundance is a highly directed process rather than a general host response to infection.

Conclusions/significance: Altered levels of miR-17 approximately 92 and miR-106b approximately 25 are known to play crucial roles in mammalian cell regulation and have been implicated in numerous hyperproliferative diseases although the mechanisms driving their altered expression are unknown. Hence, in addition to the implications of these findings on the host-pathogen interaction, Toxoplasma may represent a powerful probe for understanding the normal mechanisms that regulate the levels of key host miRNAs.

pone-0008742-g003: Neospora infection does not alter the levels of miR-17 family members.(A) Total RNA samples were extracted from HFFs during a 24-hour timecourse of Neospora-infection, and subject to primer extension analysis. From left to right, template RNAs were derived from uninfected HFFs at 6, 12 or 24 hours, or from infected HFFs at the same time points. Shown are representative autoradiographs for primer-extension with the miR-17 and 5S probes; reactions were performed on biological triplicates. miR-17 and 5S probes were also extended in the absence of template RNA (‘- RNA’ lanes) to identify any primer-extension artifacts associated with each probe. Primer-extension products are shown with arrows and labeled. Excess free probe is indicated. The size marker is a 10bp ladder. (B) A graphical summary of the primer-extension data in (A); the scale has been preserved for comparison with Figure 2B. RNA samples were subject to primer-extension with the miR-17 probe and band-intensities were quantified by phosphorimager analysis. These values were normalized to values obtained from an independent primer-extension of each RNA sample with a 5S rRNA probe. The 5S rRNA-normalized infected/uninfected ratios for each timepoint are plotted and error bars are the standard deviation between three biological replicates that were performed for each timepoint.

Mentions:
To investigate whether the observed increase in host miR-17 levels is a specific response to Toxoplasma infection or a general response of apicomplexan-infected host cells, we assayed the levels of host miR-17 in HFFs infected with Neospora caninum. Neospora and Toxoplasma are very closely related coccidians, have similar asexual growth cycles, and are morphologically almost indistinguishable [35]. Although Neospora is not a human pathogen and has a far more restricted host range than Toxoplasma, Neospora readily infects HFFs and grows with kinetics comparable to Toxoplasma in these cells [36]. To assess whether Neospora infection changes the levels of host miR-17 family in a manner similar to Toxoplasma infection, we performed primer-extension analysis on RNA samples derived from HFFs comparably infected with Neospora. The results showed that Neospora infection did not significantly alter the levels of host miR-17 family members using either a miR-17 probe (Figure 3A and Figure 3B), or miR-20 probe (data not shown) at any time-point in our assay. We conclude that the increased level of miR-17 family members seen in Toxoplasma-infected cells is Toxoplasma-specific and is not a general host response to apicomplexan infection.

pone-0008742-g003: Neospora infection does not alter the levels of miR-17 family members.(A) Total RNA samples were extracted from HFFs during a 24-hour timecourse of Neospora-infection, and subject to primer extension analysis. From left to right, template RNAs were derived from uninfected HFFs at 6, 12 or 24 hours, or from infected HFFs at the same time points. Shown are representative autoradiographs for primer-extension with the miR-17 and 5S probes; reactions were performed on biological triplicates. miR-17 and 5S probes were also extended in the absence of template RNA (‘- RNA’ lanes) to identify any primer-extension artifacts associated with each probe. Primer-extension products are shown with arrows and labeled. Excess free probe is indicated. The size marker is a 10bp ladder. (B) A graphical summary of the primer-extension data in (A); the scale has been preserved for comparison with Figure 2B. RNA samples were subject to primer-extension with the miR-17 probe and band-intensities were quantified by phosphorimager analysis. These values were normalized to values obtained from an independent primer-extension of each RNA sample with a 5S rRNA probe. The 5S rRNA-normalized infected/uninfected ratios for each timepoint are plotted and error bars are the standard deviation between three biological replicates that were performed for each timepoint.

Mentions:
To investigate whether the observed increase in host miR-17 levels is a specific response to Toxoplasma infection or a general response of apicomplexan-infected host cells, we assayed the levels of host miR-17 in HFFs infected with Neospora caninum. Neospora and Toxoplasma are very closely related coccidians, have similar asexual growth cycles, and are morphologically almost indistinguishable [35]. Although Neospora is not a human pathogen and has a far more restricted host range than Toxoplasma, Neospora readily infects HFFs and grows with kinetics comparable to Toxoplasma in these cells [36]. To assess whether Neospora infection changes the levels of host miR-17 family in a manner similar to Toxoplasma infection, we performed primer-extension analysis on RNA samples derived from HFFs comparably infected with Neospora. The results showed that Neospora infection did not significantly alter the levels of host miR-17 family members using either a miR-17 probe (Figure 3A and Figure 3B), or miR-20 probe (data not shown) at any time-point in our assay. We conclude that the increased level of miR-17 family members seen in Toxoplasma-infected cells is Toxoplasma-specific and is not a general host response to apicomplexan infection.

Bottom Line:
We show that both the primary transcripts for miR-17 approximately 92 and miR-106b approximately 25 and the pivotal miRNAs that are derived from miR-17 approximately 92 display increased abundance in Toxoplasma-infected primary human cells; a Toxoplasma-dependent up-regulation of the miR-17 approximately 92 promoter is at least partly responsible for this increase.Altered levels of miR-17 approximately 92 and miR-106b approximately 25 are known to play crucial roles in mammalian cell regulation and have been implicated in numerous hyperproliferative diseases although the mechanisms driving their altered expression are unknown.Hence, in addition to the implications of these findings on the host-pathogen interaction, Toxoplasma may represent a powerful probe for understanding the normal mechanisms that regulate the levels of key host miRNAs.

Affiliation:
Department of Microbiology and Immunology, Stanford University School of Medicine, Stanford, California, United States of America.

ABSTRACT

Background: The apicomplexan parasite Toxoplasma gondii can infect and replicate in virtually any nucleated cell in many species of warm-blooded animals; thus, it has evolved the ability to exploit well-conserved biological processes common to its diverse hosts. Here we have investigated whether Toxoplasma modulates the levels of host microRNAs (miRNAs) during infection.

Methodology/principal findings: Using microarray profiling and a combination of conventional molecular approaches we report that Toxoplasma specifically modulates the expression of important host microRNAs during infection. We show that both the primary transcripts for miR-17 approximately 92 and miR-106b approximately 25 and the pivotal miRNAs that are derived from miR-17 approximately 92 display increased abundance in Toxoplasma-infected primary human cells; a Toxoplasma-dependent up-regulation of the miR-17 approximately 92 promoter is at least partly responsible for this increase. The abundance of mature miR-17 family members, which are derived from these two miRNA clusters, remains unchanged in host cells infected with the closely related apicomplexan Neospora caninum; thus, the Toxoplasma-induced increase in their abundance is a highly directed process rather than a general host response to infection.

Conclusions/significance: Altered levels of miR-17 approximately 92 and miR-106b approximately 25 are known to play crucial roles in mammalian cell regulation and have been implicated in numerous hyperproliferative diseases although the mechanisms driving their altered expression are unknown. Hence, in addition to the implications of these findings on the host-pathogen interaction, Toxoplasma may represent a powerful probe for understanding the normal mechanisms that regulate the levels of key host miRNAs.